Development and application of an enzyme-linked immunosorbent assay (ELISA) using a soluble recombinant nucleoprotein for the detection of antibodies to avian influenza virus

ABSTRACT The unprecedented spread of highly pathogenic avian influenza virus subtype H5N1 in Asia and Europe is threatening animals and public health systems. Effective diagnosis and control management are needed to control the disease. To this end, we developed a panel of monoclonal antibodies (MAbs) against the H5N1 avian influenza virus (AIV) and implemented an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) to detect the H5 viral antigen. Mice immunized with denatured hemagglutinin (HA) from A/goose/Guangdong/97 (H5N1) expressed in bacteria or immunized with concentrated H5N2 virus yielded a panel of hybridomas secreting MAbs specific for influenza virus HA. The reactivity of each MAb with several subtypes of influenza virus revealed that hybridomas 3D4 and 8B6 specifically recognized H5 HA. Therefore, purified antibodies from hybridomas 3D4 and 8B6, which secrete immunoglobulin G (IgG) and IgM, respectively, were used as the capture antibodies and pooled hyperimmune guinea pig serum IgG served as the detector antibody. The specificity of the optimized AC-ELISA was evaluated by using AIV subtypes H5 H3, H4, H7, H9, and H10. Specimens containing AIV subtype H5 subtype yielded a specific and strong signal above the background, whereas specimens containing all other subtypes yielded background signals. The detection limits of the AC-ELISA were 62.5 ng of bacterium-expressed H5N1 HA1 protein and 124, 62, and 31 50% tissue culture infective doses of influenza virus subtypes H5N1/PR8, H5N2, and H5N3, respectively. Reconstituted clinical samples consisting of H5 AIVs mixed with pharyngeal-tracheal mucus from healthy chickens also yielded positive signals in the AC-ELISA, and the results were confirmed by reverse transcription-PCR. The tracheal swab samples from H9N2-infected chickens did not give positive signals. Taken together, the newly developed MAb-based AC-ELISA offers an attractive alternative to other diagnostic approaches for the specific detection of H5 AIV.

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An enzyme-linked immunosorbent assay for detection of avian influenza virus subtypes H5 and H7 antibodies

Acta Veterinaria Scandinavica ◽

10.1186/1751-0147-55-84 ◽

2013 ◽

Vol 55 (1) ◽

Cited By ~ 11

Author(s):

Trine H Jensen ◽

Gitte Ajjouri ◽

Kurt J Handberg ◽

Marek J Slomka ◽

Vivien J Coward ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Avian Influenza Virus ◽

Immunosorbent Assay ◽

Enzyme Linked Immunosorbent Assay

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Development of Enzyme-Linked Immunosorbent Assay with Nucleoprotein as Antigen for Detection of Antibodies to Avian Influenza Virus

Avian Diseases ◽

10.1637/7226-062204r ◽

2004 ◽

Vol 48 (4) ◽

pp. 870-878 ◽

Cited By ~ 31

Author(s):

Meilin Jin ◽

Guihua Wang ◽

Ruihua Zhang ◽

Siting Zhao ◽

Hongchao Li ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Avian Influenza Virus ◽

Immunosorbent Assay ◽

Enzyme Linked Immunosorbent Assay

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Development and evaluation of a monoclonal antibody-based competitive ELISA for the detection of antibodies against H7 avian influenza virus

BMC Veterinary Research ◽

10.1186/s12917-021-02772-6 ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Yuan Li ◽

Hongliu Ye ◽

Meng Liu ◽

Suquan Song ◽

Jin Chen ◽

...

Keyword(s):

Monoclonal Antibody ◽

Influenza Virus ◽

Avian Influenza ◽

Avian Influenza Virus ◽

Large Scale ◽

Operation Time ◽

Reference Method ◽

Antibody Detection ◽

Enzyme Linked Immunosorbent Assay ◽

Antibody Concentration

Abstract Background H7 subtype avian influenza has caused great concern in the global poultry industry and public health. The conventional serological subtype-specific diagnostics is implemented by hemagglutination inhibition (HI) assay despite lengthy operation time. In this study, an efficient, rapid and high-throughput competitive enzyme-linked immunosorbent assay (cELISA) was developed for detection of antibodies against H7 avian influenza virus (AIV) based on a novel monoclonal antibody specific to the hemagglutinin (HA) protein of H7 AIV. Results The reaction parameters including antigen coating concentration, monoclonal antibody concentration and serum dilution ratio were optimized for H7 antibody detection. The specificity of the cELISA was tested using antisera against H1 ~ H9, H11 ~ H14 AIVs and other avian viruses. The selected cut-off values of inhibition rates for chicken, duck and peacock sera were 30.11, 26.85 and 45.66% by receiver-operating characteristic (ROC) curve analysis, respectively. With HI test as the reference method, the minimum detection limits for chicken, duck and peacock positive serum reached 20, 21 and 2− 1 HI titer, respectively. Compared to HI test, the diagnostic accuracy reached 100, 98.6, and 99.3% for chicken, duck and peacock by testing a total of 400 clinical serum samples, respectively. Conclusions In summary, the cELISA assay developed in this study provided a reliable, specific, sensitive and species-independent serological technique for rapid detection of H7 antibody, which was applicable for large-scale serological surveillance and vaccination efficacy evaluation programs.

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